Researchers and industrial scientists have a need to analyze cells frown mixed populations of microorganisms for Gram sign. Unfortunately, the known methods for determination of Gram sign involve the use of organic solvents, chemical fixatives (such as formaldehyde) and staining buffers containing chelating or permeabilizing agents, each of which is known to significantly affect the properties of the cell membrane and to potentially effect cell viability.
The differentiation of Gram-positive bacteria and Gram-negative bacteria has conventionally been accomplished with a multistep colorimetric staining protocol using crystal violet and a safranin O counterstain on heat fixed bacteria. This protocol typically kills the cells being tested. In U.S. Pat. No. 4,225,669, Melnick et al. disclose a method for staining suspended bacteria using a chelating agent and a nonfluorescent dye followed by an acid wash. This method also interferes with simultaneous or subsequent analysis of viability.
Similar methods using fluorescent dyes have also been developed to take advantage of the superior sensitivity afforded by fluorescence. The fluorescent dye, rhodamine 123, was observed by Matsuyama (FEMS MICROBIOLOGY LETTERS 21, 153 (1984)) to display a slight selectivity for Gram-positive bacteria (12 Gram-positive strains versus seven of 14 Gram-negative strains). U.S. Pat. No. 4,126,516 to Messing et al. discloses a method that involves culturing the microorganisms in a fluorescent lipophilic compound that is incorporated to a greater extent in the membranes of Gram negative cells, which thus become relatively more fluorescent. These methods, however, are less accurate than conventional procedures and are more labor intensive and time consuming.
The use of special staining buffers has been found to enhance the ability of several fluorescent nucleic acid dyes to stain microorganisms. Govorunov et al. (MICROBIOLOGY 51,587 (1982)) reported that the nucleic acid stain ethidium bromide is impermeant to Gram-negative E. coli until treated with a chelating agent such as ethylenediamine tetraacetic acid (EDTA). U.S. Pat. No. 4,508,821 to Mansour et al. (1985) uses an aqueous staining buffer comprising EDTA, as well as sodium borate, formaldehyde, and a surface active agent such as Triton X-100 to detect bacteria associated with white blood cells. Likewise, U.S. Pat. No. 4,665,024 to Mansour, et al. (1987) describes the use of ethidium bromide in combination with acridine orange or thioflavin T to distinguish Gram positive and Gram negative microorganisms, optionally using a staining buffer with EDTA as above, but the method does not reliably distinguish Pseudomonas. U.S. Pat. No. 4,639,421 to Sage (1987) discloses the use of a staining buffer containing a permeabilizing agent with the fluorescent nucleic acid dye propidium iodide in conjunction with a second fluorescent dye (acridine orange, acriflavin, quinacrine, or chrysaniline), such that Gram-positive bacteria fluoresce green and Gram-negative bacteria fluoresce orange.
None of the methods previously described for determination of Gram reaction are suitable for simultaneous or subsequent determination of viability. Separate methods using fluorescent dyes for the analysis of cell viability have been developed. Live, intact cells can be distinguished from dead cells with compromised membranes by differential staining using a cell-impermeant fluorescent dye, and a cell-permeant dye that requires an intracellular reaction for the production of fluorescence. Examples of fluorescent viability stains include fluorescein diacetate, as well as nucleic acid stains acridine orange (U.S. Pat. No. 4,190,328), calcein-AM (Ser. No. 07/783,182 (filed Oct. 26, 1991) to Haugland et al. now U.S. Pat. No. 5,314,805), DAPI and Hoechst 33342. The use of acridine orange is severely limited because of high background signal and low fluorescence enhancement upon binding to nucleic acids (about two-fold). Nucleic acids complexed with DAPI or Hoechst 33342 are only excitable with UV light, which is incompatible with some instrumentation. More importantly, the spectral properties of DAPI- or Hoechst 33342-bound DNA overlap significantly with cellular autofluorescence.
The method of the present invention provides significant advantages over conventional methods for the analysis of bacteria. This method, which allows the determination of Gram sign and cell viability either simultaneously or sequentially, is extremely sensitive, reliable and fast, requires no harsh reagents or special culturing conditions, and is applicable to a wide range of microorganisms, regardless of the source. It is useful for laboratory analysis, industrial process monitoring and environmental sampling. The method comprises a combination of one to four fluorescent dyes that determine Gram sign in mixed populations of bacteria. The dyes can be used in any combination, depending on how much is initially known about the population of microorganisms being tested.
One of the dyes of the invention, from a new family of unsymmetrical cyanine dyes, was unexpectedly found to label Gram-positive bacteria and Gram-negative bacteria, whether live or dead. Although certain unsymmetrical cyanine dyes were first described before the genetic role of nucleic acids was established (Brooker, et al., J. AM. CHEM. SOC. 64, 199 (1942)), a variety of unsymmetrical cyanine dyes have now been found to be very effective in the fluorescent staining of DNA and RNA. Patent applications have been filed on DIMERS OF UNSYMMETRICAL CYANINE DYES (Ser. No. 07/761,177 filed Sep. 16, 1991 by Yue et al.) now abandoned, UNSYMMETRICAL CYANINE DYES WITH CATIONIC SIDE CHAIN (Ser. No. 07/833,006 filed Feb. 8, 1992 by Yue, et al.), now U.S. Pat. No. 5,321,130 and DIMERS OF UNSYMMETRICAL CYANINE DYES CONTAINING PYRIDINIUM MOIETIES (filed Apr. 5, 1993 by Yue et al.) (all three patent applications incorporated by reference). U.S. Pat. Nos. 4,554,546 (to Wang, et al. 1985) and 5,057,413 (to Terstappen et al. 1991) disclose use of similar derivatives of thioflavins as nucleic acid stains. U.S. Pat. No. 4,937,198 (to Lee et al. 1990) discloses a fluorescent nucleic acid stain that preferentially stains the nucleic acids of bloodborne parasites with little staining of nucleated blood cells. Closely related lower alkyl (1-6 carbons) substituted unsymmetrical cyanine dyes, exemplified by thiazole orange, are disclosed in U.S. Pat. No. 4,883,867 to Lee et al. as having particular advantages in reticulocyte analysis.
It was found that the attachment of bulkier, cyclic structures to the parent unsymmetrical cyanine dye resulted in a number of unexpected advantages for this family of dyes. For example, although bulkier, many of the new dyes more quickly penetrate the cell membranes of a wider variety of cell types, including both Gram-positive and Gram-negative bacteria and eukaryotic cells, as described in Copending Application CYCLIC-SUBSTITUTED UNSYMMETRICAL CYANINE DYES, Ser. No. 08/090,890, filed Jul. 12, 1993, incorporated by reference. Direct comparison of the rate of uptake with known dyes such as thiazole orange and its alkylated derivatives, shows enhanced uptake of many of the new compounds (Table 5, FIG. 2). Moreover, bacteria stained with selected unsymmetrical dyes with cyclic substituents exhibit greater than tenfold more fluorescence than bacteria stained with thiazole orange (Table 3). In addition, the quantum yield of the dyes of this family are unexpectedly better than that of thiazole orange (Table 1). Furthermore, by simple synthetic modification, a family of dyes having absorption and emission spectral properties that cover most of the visible and near-infrared spectrum can be prepared. The dyes of the invention have one or more of these advantageous properties. These features overcome the limitations imposed by thiazole orange and other unsymmetrical cyanine dyes for staining the nucleic acids of living cells. The superior properties exhibited by these dyes were neither anticipated nor obvious in view of the known unsymmetrical cyanine dyes.
A second fluorescent dye used in the invention was unexpectedly discovered to selectively stain the nucleic acids of live Gram-positive bacteria as well as nucleic acids of dead Gram-positive and dead Gram-negative bacteria with a red fluorescence. Two similar dyes, ethidium bromide and propidium iodide, are known to be relatively impermeant to viable cells, see e.g. Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (5th ed. 1992) and (1989) (both editions incorporated by reference), and have been used to distinguish Gram positive and Gram negative bacteria, e.g. U.S. Pat. No. 4,665,024 to Mansour, et al. (1987) supra (ethidium bromide stained several Gram positive and one Gram negative bacterial species) and U.S. Pat. No. 4,639,421 to Sage (1987) supra (propidium iodide combined with another dye in a staining buffer used to distinguish Gram negative and Gram positive bacteria). Although propidium also contains an alkyl substituent, its substituent contains a positive charge and it is even less membrane-permeant than ethidium. The second dye of the invention contains an alkyl substituent that is much longer than the ethyl group of ethidium. It stains live Gram positive cells without the use of chelators, fixatives, or other permeabilizing reagents, but does not stain live Gram negative cells. This provides significant advantages since the permeabilizing agents compromise the membrane integrity, resulting in ambiguous results with regard to cell viability. Although the second dye used for the invention also lightly stains some Gram negative organisms when used alone, the combination of dyes, as well as experience with the technique, assures that a correct result is obtained. The synthesis of the second fluorescent dye was described in J. CHEM SOC. 3059 (1952), but the reference neither discloses nor suggests the use of this compound as a nucleic acid stain as proposed in the present invention.
The third dye used in the method is an unsymmetrical cyanine dye derivative, such as those commercially available under the trademarks YOYO.TM., TOTO.TM., TO-PRO.TM., YO-PRO.TM., POPO.TM. and BOBO.TM. from Molecular Probes, Inc., Eugene, Oregon and covered under pending patents described above. As described in Molecular Probes' catalogue, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS Set 31 (5th ed. 1992), supra, the unsymmetrical cyanine dyes are membrane-impermeant dyes that can be used for probing membrane integrity, which is related to cell viability. These dyes, however, displace both the other nucleic acid stains of the invention from a nucleic acid complex. Thus, for example, in a population containing only Gram positive organisms, dead Gram positive organisms can be distinguished from live Gram positive organisms when the third dye is combined with the second dye of the invention. Furthermore, dead Gram negative bacteria and dead Gram positive bacteria can be distinguished from live Gram negative bacteria and from live Gram positive bacteria when all three dyes of the invention are used. These unexpected advantages are neither disclosed nor obvious from the HANDBOOK reference.
The fourth dye of the invention is a fluorescent reagent that binds selectively to the surface of a bacterium. This reagent is typically a protein, such as an antibody specific for cell surface components or a lectin such as wheat germ agglutinin. Although fluorescent lectins are known to bind to the outside of Gram positive microorganisms and not Gram negative microorganisms (e.g. U.S. Pat. No. 5,137,810 to Sizemore, et al. (1992) (incorporated by reference) and Sizemore, et al., APPL. ENV. MICROBIOL. 56, 2245 (1990)), and a variety of colored and fluorescent derivatives thereof have long been known, wheat germ agglutinin alone cannot be used to distinguish between live and dead Gram positive cells. Furthermore, since Gram negative organisms, whether live are dead, would only be indicated by lack of staining, wheat germ agglutinin alone does not affirmatively indicate whether any Gram negative organisms are present, particularly if they are present in minute quantities. Moreover, the preparation of the lectin-dye solution discovered for this invention gives unexpectedly superior results, so that the dye can be used without the wash step required by the Sizemore patent. The particular combination of dyes disclosed in this invention is neither anticipated nor obvious from known applications of fluorescent lectin derivatives.